During the last decade, it has become evident that S phase progression and faithful genome inheritance strongly rely on the stability of replication forks. Eukaryotic cells actively monitor replication fork progression, and temporary fork stalling elicits an S phase checkpoint response designed to prevent replication fork collapse and concomitant DNA damage. The evolutionarily conserved minichromosome maintenance protein 10 (Mcm10) plays a key role in controlling replication fork stability. A recent genome-wide screen for factors that maintain chromosomal integrity identified Mcm10 as one of only a few replication proteins that are highly effective in preventing DNA damage. However, how Mcm10 contributes to unperturbed S phase progression has remained elusive. Our studies in S. cerevisiae suggest that Mcm10 is an integral part of the eukaryotic replication fork and coordinates DNA unwinding with DNA synthesis through a multitude of interactions with other fork members. These include the replicative helicase, DNA polymerase (pol)- 1/primase and the homo-trimeric replication clamp, proliferating cell nuclear antigen (PCNA). Thus, Mcm10 may prevent replication fork collapse indirectly by properly regulating diverse fork activities. However, recent findings from our laboratory suggest that Mcm10 may also contribute more directly to eliciting an S phase checkpoint response through the binding of the hetero-trimeric checkpoint clamp 9-1-1 (Rad9/Rad1/Hus1 in S. pombe and mammals and Ddc1/Rad17/Mec3 in S. cerevisiae). Although PCNA and the 9-1-1 checkpoint clamp are structurally related, their interaction with Mcm10 is rather distinct. Importantly, ubiquitination of Mcm10 plays a crucial role in promoting the binding to one clamp (PCNA), but not the other (9-1-1). In the first part of this grant application, we seek to continue our ongoing structure-function analysis of Mcm10 in budding yeast. During the previous funding period, we identified an evolutionarily conserved PCNA interacting protein (PIP) box in Mcm10 and demonstrated that this PIP box is required for the binding between ubiquitinated Mcm10 and PCNA in S. cerevisiae. Whereas the interaction between Mcm10 and the 9-1-1 complex partially depends on a functional PIP box, it is independent of ubiquitination. Our hypothesis is that ubiquitination provides a molecular switch to target Mcm10 to PCNA. In the second part of this application, we will start to explore the pathways that connect MCM10 to the cellular genome integrity network. To this end, we have conducted a genome-wide synthetic lethality screen with the temperature sensitive mcm10-1 mutant. The top hits of this screen will be validated and further examined. The overall goal of this application is to gain a mechanistic understanding of how Mcm10 promotes the maintenance of genome stability and how cells counteract DNA damage at Mcm10-defective forks. These studies should ultimately help to dissect the molecular events that can cause genomic alterations, which contribute to tumorigenesis in humans.